Cytidine derivative dimers and applications thereof

ABSTRACT

The present disclosure provides cytidine derivative dimers, salts and compositions of the cytidine derivative dimers, and methods of making and using the cytidine derivative dimers that are useful for treating a neoplasm in mammalian subjects. A cytidine derivative dimer may have the following general formula (I): 
     
       
         
         
             
             
         
       
     
     By molecularly designing such cytidine-based compounds, the disclosed cytidine-based derivative dimers/salts show significant inhibiting effects on HCT-116 human colon cancer cells, and exhibit strong growth inhibiting effects on HCT-116 human colon cancer xenografts grown in nude mice. The disclosed cytidine derivative dimers/salts provide high anti-tumor activity with low toxicity and are useful for treating cancers.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/624,513, filed on Feb. 17, 2015. U.S. patentapplication Ser. No. 14/624,513 claims the priority of Chinese PatentApplication No. 201410652724.4, entitled “Cytidine Derivative Dimer andApplications thereof”, filed on Nov. 17, 2014, the entire content ofwhich is incorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to the field of antitumor compounds and,more particularly, relates to cytidine derivative dimers, salts andcompositions of the cytidine derivative dimers, and methods of makingand using the cytidine derivative dimers for treating various cancers.

BACKGROUND

Cancer is one of the common diseases that threaten human health. Themortality rate of cancer ranks the highest among other diseases.Currently clinical antitumor drugs face prominent toxicity issues inchemotherapy. Nowadays, an important topic on antitumor drugs is toimprove the therapeutic effect while reducing the toxicity of the drugsat the same time.

Existing cytidine compounds are mainly used for treating blood tumors.Certain cytidine compounds are also used for solid tumors. However,problems arise due to high toxicity, narrow scope of application andpoor therapeutic effect of these compounds. Further, human cancer isprone to producing drug resistance to existing cytidine compounds,resulting in failure of treatment and tumor recurrence.

The disclosed method and system are directed to solve one or moreproblems set forth above and other problems.

BRIEF SUMMARY OF THE DISCLOSURE

The technical problem to be solved by the present disclosure is toprovide a series of cytidine derivative dimers and applications thereofwith high efficacy, high anti-tumor activity and low toxicity fortreatment of various types of cancers.

One aspect of the present disclosure provides a cytidine derivativedimer of formula (I):

R1 in formula (I) is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl,—(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, where n is an integer from0 to 10, and Ph is phenyl. A carbon chain of the C₁-C₁₀ substitutedalkyl is independently substituted by a substituent selected from thegroup consisting of halogen, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof. A carbonchain or a phenyl ring of the substituted —(CH₂)_(n)-Ph (where n is aninteger from 0 to 10) is independently substituted by a substituentselected from the group consisting of halogen, cyano group, nitro group,amino group, hydroxyl group, carboxyl group, and a combination thereof.

R2 in formula (I) is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl,—(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, where n is an integer from0 to 10, and Ph is phenyl. A carbon chain of the C₁-C₁₀ substitutedalkyl is independently substituted by a substituent selected from thegroup consisting of halogen, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof. A carbonchain or a phenyl ring of the substituted —(CH₂)_(n)-Ph is independentlysubstituted by a substituent selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup, and a combination thereof.

R3 in formula (I) is hydrogen, alkoxycarbonyl or substitutedalkoxycarbonyl, wherein a substituent of the substituted alkoxycarbonylis selected from the group consisting of halogen, cyano group, nitrogroup, amino group, hydroxyl group, carboxyl group and a combinationthereof.

R4 in formula (I) is hydrogen, alkoxycarbonyl or substitutedalkoxycarbonyl, wherein a substituent of the substituted alkoxycarbonylis selected from the group consisting of halogen, cyano group, nitrogroup, amino group, hydroxyl group, carboxyl group and a combinationthereof.

R5 in formula (I) is —(CH₂)_(n)—, n being an integer from 1 to 15; orsubstituted —(CH₂)_(n)— with a substituent on a carbon chain thereof, nbeing an integer from 1 to 15. The substituent can be selected from thegroup consisting of phenyl group, substituted phenyl group, cyano group,nitro group, amino group, hydroxyl group, carboxyl group, and acombination thereof. Alternatively, R5 can be —(CH₂)_(n)—X₁—X₂—, whereX₁ is O or S, X₂ is —(CH₂)_(n)-Ph-, pyrimidyl, pyranyl, imidazolyl,pyrazinyl, or pyridyl, and n is an integer from 0 to 3.

Another aspect of the present disclosure provides a tumor inhibitingdrug including the disclosed cytidine derivative dimer or a salt formthereof.

Another aspect of the present disclosure provides a pharmaceuticalcomposition, including: a cytidine derivative dimer or apharmaceutically acceptable salt thereof as an active ingredient; andone or more of medicinal carriers and excipients, where the cytidinederivative dimer is represented by formula (I).

R1 in formula (I) is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl,—(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, where n is an integer from0 to 10, and Ph is phenyl. A carbon chain of the C₁-C₁₀ substitutedalkyl is independently substituted by a substituent selected from thegroup consisting of halogen, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof. A carbonchain or a phenyl ring of the substituted —(CH₂)_(n)-Ph is independentlysubstituted by a substituent selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup, and a combination thereof.

R2 in formula (I) is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl,—(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, where n is an integer from0 to 10, and Ph is phenyl. A carbon chain of the C₁-C₁₀ substitutedalkyl is independently substituted by a substituent selected from thegroup consisting of halogen, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof. A carbonchain or a phenyl ring of the substituted —(CH₂)_(n)-Ph is independentlysubstituted by a substituent selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup, and a combination thereof.

R3 in formula (I) is hydrogen, alkoxycarbonyl or substitutedalkoxycarbonyl, wherein a substituent of the substituted alkoxycarbonylis selected from the group consisting of halogen, cyano group, nitrogroup, amino group, hydroxyl group, carboxyl group and a combinationthereof.

R4 in formula (I) is hydrogen, alkoxycarbonyl or substitutedalkoxycarbonyl, wherein a substituent of the substituted alkoxycarbonylis selected from the group consisting of halogen, cyano group, nitrogroup, amino group, hydroxyl group, carboxyl group and a combinationthereof.

R5 in formula (I) is —(CH₂)_(n)—, n being an integer from 1 to 15; orsubstituted —(CH₂)_(n)— with a substituent on a carbon chain thereof, nbeing an integer from 1 to 15. The substituent can be selected from thegroup consisting of phenyl group, substituted phenyl group, cyano group,nitro group, amino group, hydroxyl group, carboxyl group, and acombination thereof. Alternatively, R5 can be —(CH₂)_(n)—X₁—X₂—, whereX₁ is O or S, X₂ is —(CH₂)_(n)-Ph-, pyrimidyl, pyranyl, imidazolyl,pyrazinyl, or pyridyl, and n is an integer from 0 to 3.

Another aspect of the present disclosure provides a method for preparinga cytidine derivative dimer as follows. A compound having a generalformula (II) is prepared first.

Further, a compound having a general formula (III) is prepared. Thecompound having the general formula (II) is mixed with sodium carbonateto add to a system including 1,4-dioxane and water. (Boc)₂O is furtheradded for a reaction. When the reaction is measured to be completed toprovide a reaction product, the reaction product is extracted andwashed, and then dried and concentrated to form a dried solid under areduced pressure.

The dried solid is added into chloroform. Pyridine and dianhydridesR₅(CO)₂O are also added into the chloroform for an overnight reaction toprovide a reaction product. The reaction product is concentrated toobtain a viscous oil. The viscous oil is purified by columnchromatography to obtain a compound with a general formula (IV).

R1 in formula (II) and (IV) is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl,—(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, where n is an integer from0 to 10, and Ph is phenyl. A carbon chain of the C₁-C₁₀ substitutedalkyl is independently substituted by a substituent selected from thegroup consisting of halogen, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof. A carbonchain or a phenyl ring of the substituted —(CH₂)_(n)-Ph is independentlysubstituted by a substituent selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup, and a combination thereof.

R2 in formula (III) is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl,—(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, where n is an integer from0 to 10, and Ph is phenyl. A carbon chain of the C₁-C₁₀ substitutedalkyl is independently substituted by a substituent selected from thegroup consisting of halogen, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof. A carbonchain or a phenyl ring of the substituted —(CH₂)_(n)-Ph is independentlysubstituted by a substituent selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup, and a combination thereof.

R5 in formula (IV) is —(CH₂)_(n)—, n being an integer from 1 to 15; orsubstituted —(CH₂)_(n)— with a substituent on a carbon chain thereof, nbeing an integer from 1 to 15. The substituent can be selected from thegroup consisting of phenyl group, substituted phenyl group, cyano group,nitro group, amino group, hydroxyl group, carboxyl group, and acombination thereof. Alternatively, R5 can be —(CH₂)_(n)—X₁—X₂—, whereX₁ is O or S, X₂ is —(CH₂)_(n)-Ph-, pyrimidyl, pyranyl, imidazolyl,pyrazinyl, or pyridyl, and n is an integer from 0 to 3.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are merely examples for illustrative purposesaccording to various disclosed embodiments and are not intended to limitthe scope of the present disclosure.

FIG. 1 illustrates an exemplary structural formula of a cytidinederivative dimer consistent with various disclosed embodiments;

FIG. 2 illustrates an exemplary synthetic route of a cytidine derivativedimer in Example 1 consistent with various disclosed embodiments;

FIG. 3 illustrates an exemplary synthetic route of a cytidine derivativedimer in Example 2 consistent with various disclosed embodiments;

FIG. 4 illustrates an exemplary synthetic route of a cytidine derivativedimer in Example 3 consistent with various disclosed embodiments;

FIG. 5 illustrates an exemplary synthetic route of a cytidine derivativedimer in Example 4 consistent with various disclosed embodiments;

FIG. 6 is a bar chart illustrating cell colony formation inhibitionrates of HCT-116 human colon cancer cells treated with four compounds atdifferent concentrations consistent with various disclosed embodiments;and

FIG. 7 is a curve chart illustrating a dose-response relationshipbetween inhibition rates and compound concentrations after HCT-116 humancolon cancer cells were treated with the four compounds consistent withvarious disclosed embodiments.

DETAILED DESCRIPTION

Reference will now be made in detail to exemplary embodiments of thedisclosure, which are illustrated in the accompanying drawings.Hereinafter, embodiments consistent with the disclosure will bedescribed with reference to drawings. Wherever possible, the samereference numbers will be used throughout the drawings to refer to thesame or like parts. It is apparent that the described embodiments aresome but not all of the embodiments of the present disclosure. Based onthe disclosed embodiment, persons of ordinary skill in the art mayderive other embodiments consistent with the present disclosure, all ofwhich are within the scope of the present disclosure.

As shown in FIG. 1, the present disclosure discloses an exemplarycytidine derivative dimer with a structural formula as formula (I):

where R1 can be alkyl containing from 1 to 10 carbon atoms (i.e., C₁-C₁₀alkyl), C₁-C₁₀ substituted alkyl, —(CH₂)_(n)-Ph (where n=0, 1, 2, 3, . .. , 10, and Ph is phenyl), or substituted —(CH₂)_(n)-Ph (where n=0, 1,2, 3, . . . , 10, and Ph is phenyl). The carbon chain of the substitutedalkyl can be independently substituted by one or two or three groupsincluding halogen, cyano group, nitro group, amino group, hydroxylgroup, or carboxyl group. The carbon chain or the phenyl ring of thesubstituted —(CH₂)_(n)-Ph can be independently substituted by one or twoor three groups including halogen, cyano group, nitro group, aminogroup, hydroxyl group, or carboxyl group.

In certain embodiments, R1 can be C₁-C₁₀ alkyl or —(CH₂)_(n)-Ph (wheren=0, 1, 2, 3, . . . , 10). For example, R1 is C₁-4 alkyl or—(CH₂)_(n)-Ph (where n=0, 1, 2, 3). In an exemplary embodiment, R1 isn-butyl or benzyl.

R2 in formula (I) can be C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl,—(CH₂)_(n)-Ph (where n=0, 1, 2, 3, . . . , 10, and Ph is phenyl), orsubstituted —(CH₂)_(n)-Ph (where n=0, 1, 2, 3, . . . , 10, and Ph isphenyl). The carbon chain of the substituted alkyl can be independentlysubstituted by one or two or three groups including halogen, cyanogroup, nitro group, amino group, hydroxyl group, or carboxyl group. Thecarbon chain or the phenyl ring of the substituted —(CH₂)_(n)-Ph can beindependently substituted by one or two or three groups includinghalogen, cyano group, nitro group, amino group, hydroxyl group, orcarboxyl group.

In certain embodiments, R2 can be C₁-C₁₀ alkyl or —(CH₂)_(n)-Ph (wheren=0, 1, 2, 3, . . . , 10). For example, R2 is C₁-4 alkyl or—(CH₂)_(n)-Ph (where n=0, 1, 2, 3). In an exemplary embodiment, R2 isn-butyl or benzyl.

In certain embodiments, R1 and R2 can be the same.

R3 in formula (I) can be hydrogen, alkoxycarbonyl, or substitutedalkoxycarbonyl. The substituent of the substituted alkoxycarbonyl caninclude, for example, halogen, cyano group, nitro group, amino group,hydroxyl group, or carboxyl group. In certain embodiments, R3 can be Hor alkoxycarbonyl. Optionally, R3 is H or n-butoxycarbonyl.

R4 in formula (I) can be hydrogen, alkoxycarbonyl, or substitutedalkoxycarbonyl. The substituent of the substituted alkoxycarbonyl caninclude, for example, halogen, cyano group, nitro group, amino group,hydroxyl group, or carboxyl group. In certain embodiments, R4 can be Hor alkoxycarbonyl. Optionally, R4 is H or n-butoxycarbonyl.

In certain embodiments, R3 and R4 can be the same.

R5 in formula (I) can be —(CH₂)_(n)—, where n is from 1 to 15.Alternatively, R5 can be —(CH₂)— with substituent(s) on the carbon chainthereof, and the substituent can include halogen, cyano group, nitrogroup, amino group, hydroxyl group, or carboxyl group. Or R5 can be—(CH₂)_(n)—X₁—X₂—, where n is 0, 1, 2, or 3; X₁ is O or S; and X₂ is—(CH₂)_(n)-Ph (n=0, 1, 2, or 3), pyrimidyl, pyranyl, or pyridyl. Incertain embodiments, R5 can be —(CH₂)— (where n is an integer from 1 to15) or —(CH₂)_(n)—X₁—X₂— (where n=0, 1, 2, 3, X₁ is O or S and X₂ isPh). For example, R5 is —(CH₂)_(n)— (where n is an integer from 1 to 5)or —(CH₂)—O-Ph-. Optionally, R5 is —(CH₂)₂— or —(CH₂)₃—.

Table 1 lists compounds of exemplary cytidine derivative dimers.However, the cytidine derivative dimers in the present disclosure arenot limited to these compounds.

TABLE 1 Compounds of exemplary cytidine derivative dimers. Compound No.Substituent group(s) 101 (D1) R1 and R2 are n-butyl, R3 and R4 aren-butoxycarbonyl, R5 is —(CH₂)₃— 102 R1 and R2 are n-butyl, R3 and R4are n-butoxycarbonyl, R5 is —CH₂— 103 R1 and R2 are n-butyl, R3 and R4are n-butoxycarbonyl, R5 is —(CH₂)₂— 104 R1 and R2 are n-butyl, R3 andR4 are n-butoxycarbonyl, R5 is —(CH₂)₄— 105 R1 and R2 are n-butyl, R3and R4 are n-butoxycarbonyl, R5 is —(CH₂)₅— 106 R1 and R2 aretert-butyl, R3 and R4 are n-butoxycarbonyl, R5 is —(CH₂)₃— 107 (D2) R1and R2 are n-butyl, R3 and R4 are H, R5 is —(CH₂)₃— 108 R1 and R2 aren-butyl, R3 and R4 are H, R5 is —(CH₂)₂— 109 R1 and R2 are n-butyl, R3and R4 are H, R5 is —(CH₂)₂—O—Ph— 110 R1 and R2 are n-butyl, R3 and R4are H, R5 is —(CH₂)—O—Ph— 111 R1 and R2 are tert-butyl, R3 and R4 are H,R5 is —(CH₂)₂—O—Ph— 112 (D3) R1 and R2 are benzyl, R3 and R4 are H, R5is —(CH₂)₃— 113 R1 and R2 are benzyl, R3 and R4 are H, R5 is—CHBr(CH₂)₂— 114 R1 and R2 are benzyl, R3 and R4 are H, R5 is—CHPh(CH₂)₂— 115 R1 and R2 are benzyl, R3 and R4 are H, R5 is —CHCNCH₂—116 R1 and R2 are benzyl, R3 and R4 are H, R5 is —CHBrCH₂— 117 (D4) R1is benzyl, R2 is n-butyl, R3 and R4 are H, R5 is —(CH₂)₂— 118 R1 isbenzyl, R2 is n-butyl, R3 and R4 are H, R5 is —(CH₂)₃— 119 R1 is benzyl,R2 is n-butyl, R3 and R4 are H, R5 is —(CH₂)₂—O—Ph— 120 R1 is benzyl, R2is n-butyl, R3 and R4 are H, R5 is —(CH₂)₂—O—Ph—, the para posistion ofthe phenyl ring is substituted by nitro.

When preparing the compounds in Table 1, the solid reagents employed inthe synthesis process are used directly without further treatment, theliquid reagents are used after redistilled and dried.

Example 1: Synthesis of Cytidine Derivative Dimer D1

The exemplary cytidine derivative dimer D1 can be1,5-di-[4-N-(n-butyloxycarbonyl)-3′-O-(n-butoxycarbonyl)-2′-deoxy-2′,2′-difluoro-cytidine]glutarate(also see No. 101 in Table 1) with the following structural formula:

FIG. 2 illustrates an exemplary synthetic route of D1.

In an exemplary preparation of D1, 2′-deoxy-2′, 2′-difluoro-cytidinehydrochloride (e.g., about 3 g, 10 mmol) and imidazole (e.g., about0.875 g, 12.8 mmol) in anhydrous pyridine (e.g., about 10 mL) at 0° C.were added tert-butyldimethylsilyl chloride (TBSCL) (e.g., about 3.3 g,21 mmol). The mixture was stirred for half an hour and warmed to roomtemperature, and the stirring was continued at room temperature forabout 12 hours. The mixture was treated with methanol (e.g., about 8.0mL). After stirring for about 60 minutes, the solvent was removed underreduced pressure. Compound 2 shown in FIG. 2 is then obtained in thereaction system.

Dichloromethane (DCM) (e.g., about 50 mL) and pyridine (e.g., about 10mL) were then added into the reaction system, followed by adding Butylchloroformate (e.g., about 5.46 g, 40 mmol) therein using an ice bathunder nitrogen protection. Such solution was then stirred for about 12hours at room temperature and all volatiles were removed using a rotaryevaporator, leaving settled solid. The settled solid was dissolved inethyl acetate (e.g., about 100 mL) and washed by cooled saturated sodiumbicarbonate solution (e.g., about 30 mL for two times) and brine (e.g.,about 30 mL). The resulting solution was dried over anhydrous sodiumsulfate for about 3 hours and then filtered. The filtrate was purifiedby column chromatography (e.g., dichloromethane/methanol 40:1) to obtainintermediate Compound 3 (e.g., about 3.6 grams having a two-step yieldof about 62%) as shown in FIG. 2.

Compound 3 (e.g., about 3.6 g, 6.23 mmol) was added into tetrahydrofuran(THF) (e.g., about 40 mL) and cooled in an ice bath to about 0° C.Triethylamine trihydrofluoride (e.g., about 4 mL) was slowly added.After 24 hours, the solvent was stripped to yield an orange solid, whichwas purified by column chromatography (dichloromethane/methanol, 20:1)to afford intermediate Compound 4 (e.g., about 1.68 g, with 58% yield).

The resulting intermediate Compound 4 (e.g., about 1.68 g, 3.62 mmol)was added chloroform (e.g., about 30 mL), followed by pyridine (e.g.,about 30 mL) and glutaric anhydride (e.g., about 620 mg, 5.44 mmol) inthe chloroform. The mixture was stirred at room temperature overnight.After that, 4-dimethylaminopyridine (DMAP, e.g., about 7 mg and 0.057mmol) was then added and the mixture was stirred for about 3 hours. Thereaction mixture was then concentrated to provide a viscous oilyproduct, which was purified by column chromatography to obtain Compound5 (e.g., about 1.11 g, with 53% yield).

Compound 5 (e.g., about 58 mg, 0.1 mmol), Compound 4 (e.g., about 92 mg,0.2 mmol), and N,N′-Dicyclohexylcarbodiimide (DCC, e.g., about 42 mg,0.2 mmol) were dissolved in dichloromethane (e.g., about 15 mL) andadded DMAP (e.g., about 6 mg, 0.049 mmol). After stirring for about 24hours at room temperature, dichloromethane (e.g., about 50 mL) wasadded. The mixture was then washed by water (e.g., about 10 mL),saturated brine (e.g., about 20 mL), dried over anhydrous sodiumsulfate, and concentrated in vacuo. The resulting material was purifiedby column chromatography (dichloromethane/methanol, 20:1) to affordCompound 6 (e.g., about 49 mg, with 48% yield).

The structure of Compound 6 was verified by nuclear magnetic resonance(NMR) and mass spectroscopy (MS). ¹H-NMR (MeOD-d4,400 MHz) δ: 7.97 (d,2H, J=7.68 Hz, H6-1, H6-2), 7.40 (d, 2H, J=7.68 Hz, H5-1, H5-2), 6.35(t, 2H, J=7.24 Hz, H1′-1, H1′-2), 4.47 (m, 6H, H5a′-1, H5a′-2, H5b′-1,H5b′-2, H4′-1, H4′-2), 4.21 (m, 8H, O—CH2×4), 2.53 (t, 4H, J=7.16 Hz,CH2-CH2-CH2), 1.97 (m, 2H, CH2-CH2-CH2), 1.64 (m, 8H, O—CH2-CH2×4), 1.42(m, 8H, O—CH2-CH2-CH2×4), 0.98 (m, 12H, CH2-CH3×4). ¹³C NMR (MeOD-d4,100 MHz) δ: 172.83, 164.51, 153.87, 144.53, 96.26, 77.52, 69.13, 65.89,61.94, 32.42, 30.66, 30.47, 18.83, 18.67, 12.79, 12.74, 8.48. ESIMS:calculated for C43H58F4N6O18 m/z 1023.37 (M+H)+, found 1023.66.

The synthetic route described in FIG. 2 can be adapted to producevarious cytidine derivative dimers. For example, glutaric anhydride canbe replaced with other anhydride(s) to produce corresponding compounds,such as compounds from No. 102 to No. 105 shown in Table 1. In anothercase, when tert-butyl chloroformate is used to replace butylchloroformate, the produced compound can be compound No. 106 in Table 1.

Example 2: Synthesis of Cytidine Derivative Dimer D2

The exemplary cytidine derivative dimer can be1,5-di-[4-N-(n-butoxycarbonyl)-2′-deoxy-2′,2′-difluoro-cytidine]glutarate(also see No. 107 in Table 1) with following structural formula:

FIG. 3 illustrates an exemplary reaction process. As shown in FIG. 2, anintermediate Compound 8 was prepared first. In an exemplary preparation,2′-deoxy-2′, 2′-difluoro-cytidine hydrochloride (Compound 1 shown inFIG. 3, e.g., about 300 mg, 1 mmol), Bis(trimethylsilyl)amine (HMDS)(e.g., about 5 mL, 0.023 mmol), and ammonium sulfate (e.g. about 5 mg,as catalytic) were dissolved in 1,4-dioxane (e.g., about 5 mL). Thereaction was heated under reflux for about 2 hours. Compound 19 shown inFIG. 3 was then obtained from the reaction. When the reflux reaction wascompleted, the reaction mixture was concentrated and azeotroped withtoluene twice. The resulting dried solid was dissolved indichloromethane (e.g., about 10 mL).

N-methylimidazole (e.g. about 0.24 mL, 3 mmol) and butyl chloroformate(e.g. about 0.32 mL, 3 mmol) were added into the dichloromethanesolution and the mixture was stirred at room temperature for about 4hours. The mixture was then concentrated to provide a viscous oilyproduct.

The viscous oily product was dissolved in a mixed solution oftriethylamine (e.g., about 3 mL) and methanol (e.g., about 20 mL), andstirred for about 4 hours at room temperature. Then, the solvent wasremoved under reduced pressure and the crude product was purified solvedby silica gel column chromatography with dichloromethane/methanol (e.g.,about 20:1) to afford Compound 7 (e.g., about 230 mg having a three-stepyield of about 55.5%).

The structure of Compound 7 was verified by NMR: ¹H-NMR (MeOD-d₄, 400MHz) δ: 8.30 (d, 1H, J=7.68 Hz, H6), 7.34 (d, 1H, J=7.68 Hz, H5), 6.28(t, 1H, J=7.08 Hz, H1′), 4.33 (m, 1H, H5a′), 4.0 (m, 2H, O—CH₂—CH₂—),3.81 (m, 1H, H5b′), 3.79 (m, 1H, H4′), 1.68 (m, 2H, O—CH₂—CH₂—), 1.45(m, 2H, O—CH₂—CH₂—CH₂), 0.98 (t, 3H, J=7.4 Hz, —CH₂—CH₃). ¹³C-NMR(MeOD-d₄, 100 MHz) δ 164.28, 156.27, 153.50, 144.39, 128.33, 122.72,95.81, 84.90, 81.71, 74.87, 68.88, 63.69, 59.15, 30.66, 32.40, 18.81,11.23, 8.06.

Compound 7 (e.g. about 60 mg, 0.16 mmol) and sodium carbonate (e.g.about 106 mg, 1 mmol) were dissolved in a solution of 1,4-dioxane andwater (e.g., about 5 mL, volume ratio about 4:1). Di-tert-butyldicarbonate (Boc)₂O (e.g., about 44 mg, 0.2 mmol) was added and themixture was stirred at room temperature for about 48 hours. The reactionmixture was diluted with water (e.g., about 2 mL) and extracted withethyl acetate twice (e.g., about 30 mL×2). The combined organic phasewas washed by water (e.g., about 5 mL), saturated saline (e.g., about 5mL), dried over anhydrous sodium sulfate, and then concentrated underreduced pressure. The resulting dried solid was purified by silica gelcolumn chromatography with dichloromethane/acetone/methanol (e.g., about1:1:0.02) to afford Compound 8 (e.g., about 51 mg, with 76% yield).

Referring back to FIG. 3, in an exemplary preparation of D2, to asolution of Compound 8 (e.g., about 223 mg, 0.25 mmol) in chloroform(e.g., about 6 mL) was added pyridine (e.g., about 5 mL) and butanedioicanhydride (e.g., about 100 mg, 1 mmol). The mixture was stirred at about45° C. overnight. Then the reaction mixture was concentrated to viscousoil and purified by column chromatography (e.g., DCM-MeOH about 20:1 to10:1) to afford Compound 9 (e.g. about 211 mg, with 75% yield).

Compound 9 (e.g., about 56 mg, 0.1 mmol), Compound 8 (e.g., about 92 mg,0.2 mmol) and DCC (e.g., about 42 mg, 0.2 mmol), were dissolved indichloromethane (e.g., about 15 mL) and DMAP (e.g., about 6 mg, 0.049mmol) was added. After stirring at room temperature for about 24 hours,the reaction mixture was diluted with dichloromethane (e.g., about 50mL) and washed with water (e.g., about 10 mL), saturated brine (e.g.,about 20 mL), dried over anhydrous sodium sulfate, and concentrated. Theresulting material was added trifluoroacetic acid (TFA) (e.g., about 5mL) and DCM (e.g., about 10 mL) and stirred at room temperature forabout half an hour. The reaction mixture was cooled to 0° C. andfiltered to remove any precipitate. The filtrate was concentrated andpurified by column chromatography (e.g., DCM-MeOH about 20:1 to 10:1) toafford the cytidine derivative dimer D2 (e.g., about 30 mg, with 35%yield).

The structure of D2 was verified by NMR and MS: ¹H-NMR (MeOD-d₄, 400MHz) δ 7.85 (d, 2H, J=7.68 Hz, H6-1, H6-2), 7.37 (d, 2H, J=7.68 Hz,H5-1, H5-2), 6.26 (t, 2H, J=7.24 Hz, H1′-1, H1′-2), 4.53 (m, 2H, H5a′-1,H5a′-2), 4.40 (m, 4H, H5b′-1, H5b′-2, H4′-1, H4′-2), 4.20 (m, 2H, H3-1,H3-2), 2.73 (m, 4H, —CH2-CH2-), 1.64 (m, 4H, O—CH2-CH2), 1.37 (m, 4H,O—CH2-CH2-CH2), 0.93 (m, 6H, CH2-CH3). ¹³C-NMR (MeOD-d₄, 100 MHz) δ172.41, 164.01, 155.95, 153.52, 144.36, 96.56, 70.53, 66.29, 62.49,30.74, 28.76, 19.01, 13.49. ESIMS: calculated for C₃₂H₄₀F₄N₆O₁₄ m/z809.25 (M+H)⁺, found 809.34.

The exemplary preparation described in FIG. 3 can be adapted to producevarious cytidine derivative dimers. For example, Compound 8 can reactwith other dianhydride(s), such as glutaric anhydride, to producecorresponding compounds, such as compounds from No. 108 to No. 110 shownin Table 1. In another case, when tert-butyl chloroformate is used toreplace butyl chloroformate, the produced compound can be compound No.111 shown in Table 1.

Example 3: Synthesis of Cytidine Derivative Dimer D3

The exemplary cytidine derivative dimer D3 can be1,5-di-[4-N-(benzyloxycarbonyl)-2′-deoxy-2′,2′-difluoro-cytidine]glutarate(also see No. 112 in Table 1).

FIG. 4 illustrates an exemplary synthetic route of D3.

In an exemplary preparation, Compound 13 was prepared first as follows.2′-deoxy-2′, 2′-difluoro-cytidine hydrochloride (e.g., about 300 mg, 1mmol), Bis(trimethylsilyl)amine (e.g., about 5 mL, 0.023 mmol), andammonium sulfate (e.g., about 5 mg as catalytic) were dissolved in1,4-dioxane (e.g., about 5 mL). The reaction was heated under reflux forabout 2 hours. After that, the reaction mixture was concentrated andazeotroped with toluene twice. The resulting solid was dissolved indichloromethane (e.g., about 10 mL).

N-methylimidazole (e.g. about 0.24 mL, 3 mmol) and benzyl chloroformate(e.g., about 340 mg, 3 mmol) were added to the dichloromethane solution.After stirring at room temperature for about 4 hours, Compound 20 asshown in FIG. 4 was obtained. The reaction mixture was then concentratedto provide a viscous oily product.

The viscous oily product was dissolved in a mixed solution oftriethylamine (e.g., about 3 mL) and methanol (e.g., about 20 mL), andstirred at room temperature overnight. The solvent was then removed bydistilling under reduced pressure. The crude product was purified bysilica gel column chromatography with dichloromethane/methanol (e.g.,about 20:1) to afford Compound 12 (e.g., about 162 mg, three-step yieldof about 41%).

The NMR characterizations of Compound 12 include: ¹H-NMR (MeOD-d₄, 400MHz) δ: 8.31 (d, 1H, J=7.64 Hz, H6), 7.39 (m, 5H, J=7.68 Hz, Ph), 6.25(t, 1H, J=7.12 Hz, H1′), 5.21 (s, 2H. CH₂-Ph), 4.31 (m, 1H, H5a′), 3.82(m, 2H, H5b, H4′), 3.79 (m, 1H, H3′). ¹³C-NMR (MeOD-d₄, 100 MHz) δ:164.22, 156.22, 153.27, 144.48, 135.87, 128.42, 128.10, 125.31, 122.74,120.16, 95.89, 85.35, 84.91, 81.7, 81.66, 68.87, 67.54, 58.31.

Compound 12 (e.g., about 80 mg, 0.2 mmol) and sodium carbonate (e.g.,about 106 mg, 1 mmol) were dissolved in a solution of 1,4-dioxane andwater (e.g., about 5 mL, volume ratio about 4:1). Then (Boc)₂O (e.g.,about 44 mg, 0.2 mmol) was added and the mixture was stirred for about48 hours at room temperature. After that, the reaction mixture wasdiluted with water (e.g., about 2 mL) and extracted with ethyl acetatetwice (e.g., about 30 mL X₂). The combined organic extract was washedwith water (e.g., about 5 mL), saturated saline (e.g., about 5 mL),dried over anhydrous sodium sulfate, and concentrated. The remainingresidue was then purified by column chromatography(dichloromethane/acetone/ethanol, about 1:1:0.02) to afford Compound 13(e.g., about 64 mg, with 64% yield).

To a solution of Compound 13 (e.g., about 248 mg, 0.5 mmol) inchloroform (e.g., about 15 mL) was added pyridine (e.g., about 5 mL),and glutaric anhydride (e.g., about 100 mg, 1 mmol), After stirring atabout 45° C. overnight, the reaction mixture was concentrated to viscousoil, followed by purification with column chromatography (DCM-MeOH 20:1to 10:1) to afford Compound 14 (e.g., about 223 mg, with 73% yield).

Compound 14 (e.g., about 61 mg, 0.1 mmol), Compound 13 (e.g., about 99mg, 0.2 mmol) and DCC (e.g., about 42 mg, 0.2 mmol) were dissolved indichloromethane (e.g., about 15 mL), and DMAP (e.g., about 6 mg, 0.049mmol) was added. After stirring at room temperature for about 24 hours,the reaction mixture was diluted with dichloromethane (e.g., about 50mL) and washed with water (e.g., about 10 mL), saturated brine (e.g.,about 20 mL), dried over anhydrous sodium sulfate, and concentrated. Theremaining residue was treated with TFA (e.g., about 5 mL) and DCM (e.g.,about 10 mL), followed by stirring at room temperature for about half anhour. After that, the reaction mixture was cooled in an ice-bath, andthe resulting precipitate was removed by filtration. The filtrate wasthen concentrated to provide a viscous oil, which was purified by columnchromatography (DCM-MeOH about 20:1 to 10:1) to afford D3 (e.g., about30 mg, with 35% yield).

The characterizations of D3 include: ¹H-NMR (MeOD-d₄, 400 MHz) δ: 8.31(d, 1H, J=7.64 Hz, H6), 7.39 (m, 5H, J=7.68 Hz, Ph), 6.27 (t, 2H, J=7.8Hz, H1′-1, H1′-2), 5.17 (s, 4H, CH₂-Ph×2), 4.46 (m, 4H, H5a′-1, H5a′-2,H5b′-1, H5b′-2), 4.21 (m, 2H, H4′-1, H4′-2), 4.10 (m, 2H, H3′-1, H3′-2),2.53 (t, 4H, J=7.16 Hz, —CH2-CH2-CH2-), 1.99 (q, 2H, J=7.2 Hz,—CH2-CH2-CH2-). ¹³C NMR (MeOD-d₄, 100 MHz) δ 172.90, 164.21, 155.90,153.31, 144.25, 141.03, 135.86, 128.41, 128.28, 128.10, 124.85, 123.25,122.26, 96.14, 79.17, 74.97, 67.59, 62.06, 33.19, 32.51, 19.96. ESIMS:calculated for C₃₉H₃₈F₄N₆O₁₄ m/z 891.24 (M+H)⁺, found 891.31.

The exemplary preparation described in FIG. 4 can be adapted to producevarious cytidine derivative dimers. For example, Compound 14 can reactwith other dianhydride(s), such as COOHCHBr(CH₂)₂COOH, COOHCHPh(CH₂)₂COOH, and COOH CHCNCH₂COOH, to produce correspondingcompounds, such as compounds from No. 113 to No. 116 shown in Table 1.

Example 4: Synthesis of Cytidine Derivative Dimer D4

The exemplary cytidine derivative dimer D4 can be1-O-(4-N-(Benzyloxycarbonyl)-gemcitabine)-4-O-(4-N-(n-Butoxycarbonyl)-gemcitabine)-succinat(also see No. 117 in Table 1) with the following structural formula:

FIG. 5 illustrates an exemplary synthetic route of D4.

In an exemplary preparation, Compound 9 (e.g., about 56 mg, 0.1 mmol),Compound 13 (e.g., about 99 mg, 0.2 mmol) and DCC (e.g., about 42 mg,0.2 mmol) were dissolved in dichloromethane (e.g., about 15 mL), andDMAP (e.g., about 6 mg, 0.049 mmol) was added. After stirring at roomtemperature for about 24 hours, the reaction mixture was diluted withdichloromethane (e.g., about 50 mL) and washed with water (e.g., about10 mL), saturated brine (e.g., about 20 mL), dried over anhydrous sodiumsulfate, and concentrated. The remaining residue was treated with TFA(e.g., about 5 mL) and DCM (e.g., about 10 mL) for about half an hour atroom temperature. After that, the reaction mixture was cooled in anice-bath and the resulting precipitate was removed by filtration. Thefiltrate was concentrated to provide a viscous oil and purified bycolumn chromatography (DCM-MeOH about 20:1 to 10:1) to afford D4 (e.g.,about 30 mg, 36% yield).

The characterizations of D4 include: ¹H-NMR (MeOD-d₄, 400 MHz) δ 7.98(m, 2H, H6-1, H6-2), 7.40 (d, 2H, J=7.68 Hz, H5-1, H5-2), 7.38 (m, 6H,Ph), 6.26 (t, 2H, J=8 Hz, H1′-1, H1′-2), 5.21 (s, 2H, CH₂-Ph), 4.43 (m,2H, H5a′-1, H5a′-2), 4.29 (m, 2H, H5b′-1, H5b′-2), 4.21 (m, 6H, H4′-1,H4′-2 H3′-1, H3′-2), 2.74 (m, 4H, —CH₂—CH₂—), 1.43 (m, 2H, O—CH₂—CH₂—),1.28 (m, 2H, O—CH₂—CH₂—CH₂—), 0.97 (t, 3H, J=7.4 Hz, —CH₂—CH₃). ¹³C NMR(MeOD-d₄, 100 MHz) δ 172.56, 164.19, 155.89, 153.52, 144.61, 135.86,128.09, 122.22, 96.21, 79.38, 78.91, 70.83, 67.60, 65.92, 62.11, 56.72,30.64, 28.66, 28.51, 25.93, 18.82, 14.26, 12.77. ESIMS: calculated forC₃₅H₃₈F₄N₆O₁₄ m/z 843.24 (M+H)⁺, found 843.33.

The exemplary preparation described in FIG. 5 can be adapted to producevarious cytidine derivative dimers. For example, butanedioic anhydridecan be replaced with other anhydride(s) to react with Compound 8 andproduce an intermediate compound. The intermediate compound can thenreact with Compound 13 to produce corresponding compounds, such ascompounds from No. 118 to No. 120 shown in Table 1.

Example 5: Preparation of Cytidine Derivative Dimer Hydrochloride

In an exemplary preparation of hydrochloride of the exemplary cytidinederivative dimer in Example 1 including exemplary cytidine derivativedimer D1,1,5-di-[4-N-(n-butyloxycarbonyl)-3′-O-(n-butoxycarbonyl)-2′-deoxy-2′,2′-difluoro-cytidine]glutarate(e.g., about 0.50 g) was dissolved in ethyl acetate (e.g., about 60 mL).The solution was cooled in an ice bath and treated with dry hydrochloricacid gas. After stirring for 15 minutes, the solvent was removed toobtain the HCl salt of the product as white solid.

Similar procedure can be used to prepare other hydrochloride of thedisclosed cytidine derivative dimer(s).

In addition to hydrochloride salt disclosed herein, other cytidinederivative dimer-based salts including phosphates, sulfates, carbonates,nitrates, citrates, tartrates, maleates, succinates, sulfonates,p-toluenesulfonates, methanesulfonates, benzoates or fumarates of thedisclosed cytidine derivative dimer can also be prepared accordingly.

The present disclosure also relates to treatment of cancer. Morespecifically, various embodiments may be directed to the treatment of asubject, particularly a mammal such as a human, having a neoplasm byadministering a therapeutically effective amount of a novel compound offormula (I) to the subject for a period of time sufficient to produce ananti-neoplastic result.

The term cancer is to be considered in the broadest general definitionas a malignant neoplasm, an abnormal mass of tissue, the growth of whichexceeds and is uncoordinated with that of normal tissues and persists inthe same excessive manner after cessation of the stimuli that evoked thechange. It might be added that the abnormal mass is purposeless, preyson the host, and is virtually autonomous. A cancer can also beconsidered as a malignant tumor. Various types of cancers, i.e.,malignant tumors or neoplasia may be treated by administering thedisclosed compound.

The disclosed compounds can be useful in the treatment of a neoplasm,e.g. leukemia and solid tumors, such as colon, colo-rectal, ovarian,mammary, prostate, lung, kidney and melanoma tumors. The dosage rangeadopted may depend on the route of administration and on the age, weightand condition of the patient being treated. The compounds may beadministered, for example, by the parenteral route, for example,intramuscularly, intravenously or by bolus infusion.

As used herein, a patient or subject is a vertebrate having cancer orother diseases. Preferably, the subject is a warm-blooded animal,particularly a mammal which includes both human and non-human mammals.Examples of non-human mammals include but are not limited to farmanimals, such as cows, sheep, pigs, goats, horses, and llama, and pets,such as dogs and cats. More preferably, the subject is a human. Thecompounds are shown herein as Formula I and are described in more detailhereinafter. A therapeutically effective amount of the compound isadministered to a subject in need thereof for a period of timesufficient to obtain an antineoplastic effect.

With mammals, including humans, the effective amounts can beadministered on the basis of body surface area. A suitable dose rangemay be from 1 mg to 1000 mg of equivalent disclosed compound per m² bodysurface area, for instance from 50 mg/m² to 500 mg/m².

Example 6: Lyophilized Powder of Cytidine Derivative Dimer for Injection

In an exemplary preparation of injectable lyophilized powder of compoundD3 in Example 3, the injectable lyophilized powder of D3 may includecompound D3 (e.g. about 30 g), mannitol (e.g., 20% w/v, about 300 g),buffer sodium dihydrogen phosphate dihydrate (e.g., about 7 g), andsurfactant poloxamer 188 (F68) (e.g., about 4.0 g).

The sodium dihydrogen phosphate dihydrate, the poloxamer 188 (F68) (CASNo.: 9003-11-6), and the mannitol (e.g., about 20% w/v) were weighedaccording to the prescription amount as suggested above, and thendissolved in water (e.g., about 300 g) for injection which waspre-cooled to a temperature lower than about 10° C. NaOH (0.1 mol/L) wasused to adjust the pH value of the solution to about 7.3-7.5. Prescribeddosage of D3 was then added to the solution and homogeneously mixed.NaOH solution (0.1 mol/L) or HCl (0.1 mol/L) can be used to adjust thepH value to about 7.3±0.2 (about 7.5 in this Example). The solution wasadded with water until 2000 g, and then sterilized by 0.22 μmmicrofiltration. The filtrate was dispensed in vials for about 2.0 gramsin each vial. The vials were partially stoppered, and placed in a freezedryer for lyophilization. After drying, the vials were vacuum packed,capped, and labeled to prepare about 1000 vials of lyophilized powderinjections. The storage temperature was about 2° C.-8° C.

In addition to the injectable lyophilized powder (i.e., sterile powderfor injection), the cytidine derivative dimer(s) in present disclosuremay be formulated in any other suitable forms for injection, such as asolution-type injection, a suspension-type injection, an emulsion-typeinjection, liposomal injection, and/or a sterile powder-type forinjection.

Example 7: Pharmaceutical Composition of Cytidine Derivative Dimer(s)

An exemplary pharmaceutical composition of cytidine derivative dimer mayinclude pharmaceutically active ingredients and excipients. Thepharmaceutically active ingredient may include the disclosed cytidinederivative dimer or corresponding salt thereof. The weight percentage ofthe active ingredient in the pharmaceutical composition can be about 1%to about 95% (about 30% in this Example). The excipients may includewater, lactose, corn starch, hydroxypropylmethyl cellulose and magnesiumstearate. The pharmaceutical compositions can be in various formsincluding, for example, a tablet.

In addition to the tablet form, the pharmaceutical composition can takeother suitable forms. The pharmaceutically active ingredient can beprepared for oral administration in a form as: powders, granules,capsules, pellets formulations, solutions, suspensions, emulsions,syrups or elixirs, or slow-controlled release formulation and controlledrelease formulations, or other suitable forms for oral administration.These orally administered types of pharmaceutical composition maycontain commonly used excipients (including, e.g., additives,appendages, etc. depending on their functions) corresponding to thepharmaceutical composition. The additives may include mannitol, lactose,starch, magnesium stearate, sugar, salt, cellulose, magnesium sulfate,etc. in pharmaceutical grades.

When preparing the exemplary orally administered type of pharmaceuticalcomposition, appendages in pharmaceutical grade may be used as medicinalcarriers for carrying the pharmaceutically active ingredients andinclude, for example: inert solid diluents, aqueous solvents, liposomes,microspheres and/or non-toxic organic solvents. Any suitable appendagesin pharmaceutical grade can be used herein. In one embodiment, theappendages in pharmaceutical grade may include: wetting agents,emulsifying agents, pH buffers, human serum albumin, antioxidants,preservatives, bacteriostatic agents, dextrose, sucrose, trehalose,maltose, lecithin, glycine, sorbic acid, propylene glycol, polyethylene,protamine, boric acid, sodium chloride, potassium chloride, mineral oil,vegetable oil, or a combination thereof.

The target tumors of the disclosed pharmaceutical composition mayinclude blood tumors (e.g., neoplastic hematologic disorder) ormalignant solid tumors. Specifically, target tumors may include lungcancer, prostate cancer, breast cancer, colon cancer, stomach cancer,pancreatic cancer, liver cancer, esophageal cancer, brain tumor, ovariancancer, uterine cancer, kidney cancer, head and neck cancer, skincancer, bladder cancer, vulvar cancer, testicular tumor, colorectalcancer, choriocarcinoma, germ cell tumors, malignant lymphoma, leukemiaand multiple myeloma. Further, an exemplary target tumor may includepancreatic cancer (in a first-line or second-line treatment), non-smallcell lung cancer, small cell lung cancer, breast cancer, ovarian cancerand head and neck squamous cell carcinoma, and/or colon cancer. However,target tumors that may be treated by the disclosed pharmaceuticalcomposition are not limited thereto.

Application Example 1: Inhibition Test of a Series of Compounds onHCT-116 Human Colon Cancer Cell Colony Formation

1. The colony formation inhibition test was used to evaluate the effectof four exemplary candidate compounds (D1, D2, D3, and D4) havingconcentrations of 50 nM, 150 nM and 450 nM on inhibiting cellproliferation of HCT-116 human colon cancer cells line.

2. Experiment material includes cell lines. HCT-116 human colon cancercell line was purchased from the Chinese Academy of Sciences ShanghaiCell Resource Center, Cat # TCHu 99.

3. Reagent preparation includes HCT-116 human colon cancer cell culturemedium, e.g., Dulbecco's Modified Eagle's medium (DMEM) medium with 10%fetal bovine serum (FBS). Compound solutions were prepared by dissolvingand diluting the above synthesized compounds in dimethyl sulfoxide(DMSO) to a final concentration of about 100 μM.

Cell staining solution was prepared including 0.5% crystal violetsolution prepared using absolute or anhydrous ethanol, and stored indark. Before staining, the solution was mixed with phosphate bufferedsaline (PBS) buffer solution by volume ratio of 1:4 as the cell stainingsolution.

4. Cell culture: cells in logarithmic growth phase were collected,counted, and resuspended in complete culture medium. The cellconcentration was adjusted to an appropriate concentration, 6-wellculture plates were seeded, with about 300 cells and 1.8 mL of culturemedium in each well. Cells were incubated for about 5 hours in anincubator at about 37° C. and about 100% relative humidity and under 5%CO₂.

5. Cell colony formation inhibition test and data processing wereperformed including, for example, (a)-(i) as follows.

(a) Cells in logarithmic growth phase were collected and counted. Thecells were re-suspended in culture medium containing 5% FBS, andcounted. Six-well plates were seeded according to a rate of 300 cellsper well. Cells were incubated for about 5 hours in an incubator atabout 37° C. with 100% relative humidity and under 5% CO₂.

(b) The compounds were diluted with culture medium (containing 5% FBS)to be concentrations of about 0.5 μM, 1.5 μM and 4.5 μM. Adding thecompounds to cells in 200 μL per well, the final concentrations of thecompounds were about 50 nM, 150 nM and 450 nM. Each concentration pointwas repeatedly tested for three times.

(c) Cells were incubated for about 72 hours in an incubator at 37° C.with 100% relative humidity and under 5% CO₂. (d) The culture mediumfrom the plates (the culture medium containing the compound(s) wasaspirated. The plates were rinsed with Hank's Balance Salt Solution(HBSS) twice, and replaced with fresh culture medium (DMEM medium with15% FBS).

(e) Cells were incubated for about 7-10 days in an incubator at about37° C. and 100% relative humidity and under 5% CO₂, until the formationof visible cloned plaques. (f) The culture medium was aspirated from theplates. The plates were rinsed twice with PBS solution. (g) The residualPBS was absorbed and removed, and ethanol was added at 1 mL per plate,fixing for 30 minutes.

(h) Ethanol was absorbed and removed, and cell staining solution wasadded, stained for 3 minutes. (i) The staining solution was aspirated.After rinsing three times with PBS, the cells were counted.

Data processing was performed. Cloning formationefficiency=[As/Ac]×100%; and colony formation inhibition rate=1−cloningformation efficiency, where As denotes number of cell colonies incompound-treated group (cells+compounds to be tested), and Ac denotesnumber of cell colonies in negative control (without treating withcompound) (cells+1% DMSO).

6. Results and discussion: Table 2 lists numbers of cell colonies ofHCT-116 human colon cancer cells treated with the four compounds, where% inhibition means inhibition rate.

TABLE 2 Numbers of cell colonies of HCT-116 human colon cancer cellstreated with the four compounds. Number of cell clones on HCT-116 DG-1DG-2 DG-3 DG-4 mean % inhibition mean % inhibition mean % inhibitionmean % inhibition Control 251 255 241 256  50 nM 242 3.58% 243 4.70% 2371.66% 235 8.20% 150 nM 188 25.10% 191 25.10% 20 91.70% 75 70.70% 450 nM52 79.28% 36 85.88% 4 98.34% 11 95.70%

FIG. 6 is a bar chart illustrating the cell colony formation inhibitionrates of HCT-116 human colon cancer cells treated with the fourcompounds at concentrations of about 50 nM, 150 nM and 450 nM. Table 2and FIG. 6 show that the disclosed compounds have significant effects ininhibiting tumor cells.

FIG. 7 is a curve chart illustrating the dose-response relationshipbetween the inhibition rates and the compound concentrations when thefour compounds reacting with the HCT-116 human colon cancer cells. Asshown in FIG. 7, the IC50 value of D1 was about 245.3 nM, the IC50 valueof D2 was about 226.6 nM, the IC50 value of D3 was about 99.80 nM, andthe IC50 value of D4 was about 111.7 nM.

Application Example 2: Effects of a Series of Compounds on Tumor GrowthInhibition

By observing the tumor formation and growth at inoculation sites andchanges in body weight of test animals, this application exampleevaluated tumor growth inhibition of HCT-116 human colon cancerxenografts and toxicity of a single intraperitoneal injection of thecompounds D1 to D4 in HCT-116 colon tumor-bearing nude mice.

1. Experiment objectives were to measure tumor growth inhibition ofHCT-116 human colon cancer xenografts and to evaluate toxicity of asingle intraperitoneal injection of the disclosed cytidine derivativedimer compounds to HCT-116 colon tumor-bearing nude mice.

2. Preparation of the test substance was performed by dissolving thetest substance using the following solvents:

Solvent Lot number Suppliers Absolute ethanol 10009218 SinopharmChemical Reagent Co., Ltd (Shanghai, China) Cremophor EL 27963Sigma-Aldrich (Shanghai, China) 0.9% saline 13083004 HUA YUPharmaceutical Co., Ltd (Wuxi, China)

Corresponding test substance was weighed and put into a 5 mL glass tube.The test substance was dissolved in ethanol under the stirring by a 5 mmmagnetic stirrer. After a complete dissolution, Cremophor EL was addedwith continuous stirring. Immediately prior to the user of the testsubstance, the labeled amount of physiological saline was added andstirred. The ethanol, Cremophor EL, physiological saline had a volumeratio of 5:5:90 in a final solution.

3. Animal experiments were performed. The animals were female Balb/cnude mice at specific pathogen free (SPF) level and were supplied fromShanghai Sippr/BK Lab Animal Ltd. (Shanghai, China). For example, 40animals were obtained and ones in desired health were selectedthere-from for the experiments. All animals had a certificate ofconformity, e.g., with certificate number 0123627. These animals were atthe age of about 7 weeks to about 9 weeks at the beginning of theexperiments and had the weight of about 18 grams to about 22 grams atthe beginning of the experiment. The animals had acclimatizationduration of about 5 days to about 7 days. Animals were numbered usingtheir tail number. The animal room was maintained at about 23±2° C.,40%-70% humidity, with alternating light and dark every 12 hours.

Animal foods (SLAC-M01) were purchased from Beijing Ke Ao Xie LiCooperation Limited (Beijing, China). Sterilized filtration water wasused for the experimental animals. During the experiment period, theanimals could eat and drink freely.

4. Experimental methods were provided.

4.1 Tumor cells were human HCT-116 colon cancer cells purchased from theInstitute of Cell Biology, Chinese Academy of Sciences (Beijing, China).F-12 medium (containing 10% FBS) was used to culture cells in a carbondioxide incubator at about 37° C., saturated humidity, under 5% CO₂ and95% air. Before inoculation, cells in logarithmic growth phase werecollected. After digested by 0.25% trypsin, the cells were washed withPBS once. The cells were resuspended with serum-free medium and counted.Serum-free medium were used for cell resuspension, until the cellconcentration was adjusted to about 3×10̂7 cell/mL.

4.2 Animal inoculation and grouping were performed. Under asepticconditions, each nude mice was injected with 0.1 mL cell suspension intothe right hind leg subcutaneously (3×10̂6 cell/mouse). When the size ofthe tumor grew to have a volume of about 60-150 mm³, nude mice with asimilar tumor size and a desirable shape (e.g., with substantially sameshape such as a substantially spherical shape, without having irregularshapes or multiple tumors grouped together) were selected and grouped.In the experiment, there were six mice in each group. The groupingsituation was listed in Table 3, where IP denotes intraperitonealinjection, and “QD×1” means one injection.

TABLE 3 Animal grouping and inoculation. Group Number of DosageAdministration No. Medication animals (mg/kg) method Injection 1 D1 6400 IP QD × 1 2 D2 6 400 IP QD × 1 3 D3 6 350 IP QD × 1 4 D4 6 300 IP QD× 1 13 Control 6 — IP QD × 1

The mice in control group were injected with a mixed solution includingethanol, Cremophor EL and normal saline at a ratio of 5:5:90.

4.3 Drug administration and observation of animals were provided.

Nude mice in each group were observed for tumor formation and growth atinoculation sites. The diameters (D) of the tumor nodules were measuredthree times a week with a circle sizer ruler. In addition, the followingformula was applied to calculate the volume of the tumor nodules (V):V=3/4π(D/2)³ generally for a spherical shape. Evaluation indexes ofantitumor activity were tumor growth inhibition rate TGI (%) andrelative tumor proliferation rate T/C (%).

The calculation formula of TGI was: TGI(%)=(V_(control)−V_(Treatment))/V_(control))×100%. The relative tumorvolume (RTV) was calculated by: RTV=Vt/V0, where V0 is the tumor volumeat the time of group administration, and Vt is the tumor volume at thetime of measurement.

Relative tumor proliferation rate T/C (%) was calculated as: T/C(%)=T_(RTV)/C_(RTV)×100%. T_(RTV) denotes treatment group RTV; andC_(RTV) denotes negative control group RTV. The mice were weighed 3times every week.

4.4 Clinical symptoms were provided. All clinical symptoms of eachanimal were recorded at the beginning of the experiments and during theexperiments. The observation was performed at the same time every day.

After administration of the test substance, when the weight reductionwas >20%, when the animal was dying, or when the tumor volume exceedsabout 2800 mm³, the animal were euthanized by applying CO₂. The tumorwas separated and weighed, an autopsy was performed, and visualinspection was conducted and recorded on whether there were pathologicalchanges in the organs.

4.5 Data and statistical analysis were provided. Unless otherwiseindicated, experimental data were presented by Mean±SEM; unpaired T-testwas used for comparing between two groups, the result was consideredsignificantly different when P<0.05.

5. Experiment results included the following. (1) The influence of thetest compounds on the weight of tumor-bearing mice with human coloncancer HCT-116 was measured. The average weight of the animals in eachgroup is shown in Table 4, where “*” indicates p-value<0.05 versus thecontrol group and “**” indicates p-value<0.01 versus the control group.

TABLE 4 Average weight of the animals in each group after treatment.Dosage Groups (mg/kg) Day 0 (g) Day 1 (g) Day 2 (g) Day 4 (g) Day 7 (g)D1 400 19.98 ± 0.49 19.48 ± 0.58 17.45 ± 0.81* 16.15 ± 1.08** 19.80 ±1.40 D2 400 19.87 ± 0.25 19.85 ± 0.28 18.17 ± 0.31** 17.75 ± 0.44**18.48 ± 0.40 D3 350 19.95 ± 0.09 19.23 ± 0.31 17.70 ± 0.26** 18.24 ±0.58* 19.51 ± 0.48 D4 300 20.15 ± 0.29 19.90 ± 0.19 18.48 ± 0.2** 19.07± 0.36 19.85 ± 0.38 Control (NA) 20.55 ± 0.37 20.43 ± 0.45 20.07 ± 0.3820.02 ± 0.29 19.33 ± 0.26 Dosage Groups (mg/kg) Day 9 (g) Day 11 (g) Day14 (g) Day 16 (g) Day 18 (g) D1 400 20.75 ± 1.05 21.25 ± 1.55 21.35 ±1.35* 21.75 ± 1.25* 23.30 ± 1.60** D2 400 19.92 ± 0.45 20.42 ± 0.5320.85 ± 0.60* 21.13 ± 0.51** 21.63 ± 0.51** D3 350 20.74 ± 0.38 21.12 ±0.41 21.26 ± 0.21** 22.00 ± 0.40** 22.58 ± 0.38** D4 300 20.73 ± 0.29*21.05 ± 0.31 20.90 ± 0.34** 21.48 ± 0.34** 21.95 ± 0.27** Control (NA)19.85 ± 0.23 20.23 ± 0.25 19.42 ± 0.19 19.62 ± 0.29 19.68 ± 0.23 DosageGroups (mg/kg) Day 21 (g) Day 23 (g) Day 25 (g) Day 28 (g) Day 30 (g) D1400 22.30 ± 1.30** 22.60 ± 1.50* 23.20 ± 1.70 23.80 ± 1.50 23.60 ± 1.20D2 400 20.72 ± 0.46* 20.60 ± 0.40 20.88 ± 0.48 20.18 ± 0.44 20.33 ± 0.55D3 350 21.98 ± 0.41** 22.44 ± 0.41** 22.50 ± 0.53** 22.12 ± 0.48* 22.90± 0.60 D4 300 21.63 ± 0.30** 22.03 ± 0.43** 22.30 ± 0.49** 21.88 ± 0.5922.03 ± 0.53 Control (NA) 19.30 ± 0.24 19.55 ± 0.18 20.08 ± 0.30 19.75 ±0.05 N/A

TABLE 5 Average rate of weight change of the animals in each group aftertreatment. Dosage Groups (mg/kg) Day 0 (%) Day 1 (%) Day 2 (%) Day 4 (%)Day 7 (%) D1 400 0.00 −2.55 ± 0.77 −12.18 ± 2.63** −18.58 ± 4.66** −4.23± 3.77 D2 400 0.00 −0.08 ± 0.74  −8.54 ± 1.31** −10.63 ± 2.13** −6.98 ±1.43 D3 350 0.00 −3.59 ± 0.66 −11.25 ± 0.96**  −8.10 ± 2.84 −1.68 ± 2.50D4 300 0.00 −1.17 ± 1.27  −8.18 ± 1.56**  −5.33 ± 1.78 −1.43 ± 1.92Control (NA) 0.00 −0.60 ± 0.59  −2.35 ± 0.75  −2.55 ± 0.82 −5.85 ± 1.13Dosage Groups (mg/kg) Day 9 (%) Day 11 (%) Day 14 (%) Day 16 (%) Day 18(%) D1 400  0.42 ± 1.92  2.77 ± 4.27  3.29 ± 3.29  5.24 ± 2.74 12.70 ±4.20** D2 400  0.21 ± 1.42  2.72 ± 1.85  4.87 ± 2.08**  6.32 ± 1.65** 8.83 ± 1.43** D3 350  4.50 ± 1.93*  6.38 ± 1.68*  7.12 ± 1.19**  10.78± 0.88** 13.71 ± 0.63** D4 300  2.91 ± 0.55**  4.47 ± 0.52*  3.72 ±0.73**  6.62 ± 0.79**  8.99 ± 1.38** Control (NA) −3.30 ± 1.54 −1.39 ±2.07  −5.39 ± 1.69  −4.38 ± 2.27 −4.05 ± 2.20 Dosage Groups (mg/kg) Day21 (%) Day 23 (%) Day 25 (%) Day 28 (%) Day 30 (%) D1 400  7.90 ± 2.90* 9.32 ± 3.82*  12.20 ± 4.70  15.14 ± 3.64 14.22 ± 2.22 D2 400  4.22 ±1.23**  3.66 ± 1.05**  5.07 ± 1.44  1.57 ± 1.50  2.28 ± 1.75 D3 35010.72 ± 1.6** 13.08 ± 2.31**  13.34 ± 2.34**  11.44 ± 2.29* 15.37 ± 2.93D4 300  7.41 ± 1.37**  9.37 ± 1.82**  10.69 ± 2.14**  8.62 ± 2.68  9.36± 2.21 Control (NA) −5.94 ± 2.02 −3.59 ± 1.66  −0.97 ± 2.53  −2.68 ±5.27 N/A

As indicated by data in the Tables 4 and 5, after intraperitonealinjecting each compound into the tumor-bearing nude mice with HCT-116colon cancer xenografts, D1 group with a dose of 400 mg/kg of D1compound showed body weight was significantly decreased on Day 4 afterD1 drug administration, followed by steady weight growth in later days.After that, the weight was significantly increased comparing with thecontrol group. There were no significant differences between otherdrug-treated groups and the control group, indicating no toxicity of thethree compounds (D2, D3 and D4) in tumor-bearing mice.

(2) The effect of the test compounds on the tumor volume of HCT-116human colon cancer xenografts in nude mice was measured. Table 6 showsthe detailed data about tumor volumes of each group, where “*” indicatesp-value<0.05 versus the control group and “**” indicates p-value<0.01versus the control group.

TABLE 6 Effects of the four cytidine derivative dimers on HCT-116 humancolon cancer xenografts. Dosage Groups (mg/kg) Day 0 (mm³) Day 1 (mm³)Day 2 (mm³) Day 4 (mm³) D1 400 39.36 ± 6.16** 58.20 ± 17.45** 58.20 ±17.45** 37.24 ± 9.27** D2 400 53.09 ± 9.33** 50.78 ± 14.16** 55.89 ±18.82**  45.96 ± 20.25** D3 350 38.77 ± 4.33** 36.40 ± 3.98**  36.40 ±3.98**  23.00 ± 3.08** D4 300 44.09 ± 5.99** 52.03 ± 12.91** 52.03 ±12.91**  37.37 ± 10.46** Control (NA) 109.55 ± 8.60   180.51 ± 9.97  221.87 ± 11.44   296.98 ± 22.98  Dosage Groups (mg/kg) Day 7 (mm³) Day 9(mm³) Day 11 (mm³) Day 14 (mm³) D1 400  36.82 ± 28.63**  50.63 ± 36.49**101.02 ± 78.57** 285.66 ± 237.94* D2 400  35.87 ± 21.79**  30.75 ±16.74** 57.29 ± 33.0** 116.87 ± 66.85** D3 350 12.62 ± 3.10** 12.62 ±3.10** 14.28 ± 3.69** 36.98 ± 4.82** D4 300 25.46 ± 8.17** 22.50 ±5.31** 26.19 ± 5.79**  70.28 ± 18.37** Control (NA) 432.47 ± 39.48 590.34 ± 60.83  976.15 ± 83.07  1440.15 ± 144.73  Dosage Groups (mg/kg)Day 16 (mm³) Day 18 (mm³) Day 21 (mm³) Day 23 (mm³) D1 400 495.95 ±408.8**  647.07 ± 503.28** 1017.61 ± 749.53 1017.61 ± 749.53 D2 400169.73 ± 88.15**  262.10 ± 111.14**  489.13 ± 174.91  501.98 ± 170.25 D3350  80.10 ± 11.07** 120.40 ± 23.44** 204.05 ± 36.25 229.06 ± 36.15 D4300 142.77 ± 49.34** 228.58 ± 76.92** 375.06 ± 86.27 420.73 ± 76.08Control (NA) 1811.14 ± 119.30  1998.33 ± 136.40  2444.84 ± 167.642361.69 ± 146.79 Dosage Groups (mg/kg) Day 25 (mm³) Day 28 (mm³) Day 30(mm³) D1 400 1044.35 ± 722.80 1296.79 ± 847.87 1589.29 ± 983.15 D2 400 569.05 ± 189.18  689.78 ± 203.52  900.52 ± 250.44 D3 350 266.11 ± 39.63347.17 ± 60.84 479.01 ± 75.54 D4 300 491.29 ± 83.78 608.08 ± 85.37 781.78 ± 105.33 Control (NA) 2582.36 ± 155.95 2689.30 ± 116.86 N/A

As indicated by the detailed data of tumor volumes of each group inTable 6, the four disclosed cytidine derivative dimers significantlyinhibited the growth of HCT-116 human colon cancer xenografts.

(3) Tumor growth inhibition rate (TGI %) of the test compounds onHCT-116 human colon cancer xenografts was measured. The tumor growthinhibition rate (TGI %) of the test compounds D1-D4 on HCT-116 humancolon cancer xenografts are shown as follows in Table 7.

TABLE 7 Tumor growth inhibition rate (TGI %) after treatment with fourcytidine derivative dimers. Dosage Day 0 Day 1 Day 2 Day 4 Day 7 Group(mg/kg) (TGI %) (TGI %) (TGI %) (TGI %) (TGI %) D1 400 0.00 67.76 73.7787.46 91.49 D2 400 0.00 71.87 74.81 84.53 91.71 D3 350 0.00 79.83 83.5992.26 97.08 D4 300 0.00 71.17 76.55 87.42 94.11 Dosage Day 9 Day 11 Day14 Day 16 Day 18 Group (mg/kg) (TGI %) (TGI %) (TGI %) (TGI %) (TGI %)D1 400 91.42 89.65 80.16 72.62 67.62 D2 400 94.79 94.13 91.88 90.6386.88 D3 350 97.86 98.54 97.43 95.58 93.97 D4 300 96.19 97.32 95.1292.12 88.56 Dosage Day 21 Day 23 Day 25 Day 28 Day 30 Group (mg/kg) (TGI%) (TGI %) (TGI %) (TGI %) (TGI %) D1 400 58.38 56.91 59.56 51.78 43.36D2 400 79.99 78.74 77.96 74.35 67.91 D3 350 91.65 90.30 89.70 87.0982.93 D4 300 84.66 82.19 80.98 77.39 72.14

The tumor inhibition rate of compound D1 group with 400 mg/kg dosagereached to a maximum of 91.49% on Day 7. The tumor inhibition rate ofcompound D2 group with 400 mg/kg dosage reached to a maximum of 94.79%on Day 9. The tumor inhibition rate of compound D3 group with 350 mg/kgdosage reached to a maximum of 98.54% on Day 11. The tumor inhibitionrate of compound D4 group with 300 mg/kg dosage reached to a maximum of97.32% on Day 11.

(4) Effects of four test compounds on relative tumor volume (RTV) ofHCT-116 human colon cancer xenografts were provided.

Table 8 shows the significant effects of D1-D4 test compounds onrelative tumor volume (RTV) of HCT-116 human colon cancer xenografts,where “*” indicates p-value<0.05 versus the control group and “**”indicates p-value<0.01 versus the control group.

TABLE 8 Effects of four cytidine derivative dimers on RTV of HCT-116human colon cancer xenografts. Dosage Day 0 Day 1 Day 2 Day 4 Day 7Groups (mg/kg) (RTV) (RTV) (RTV) (RTV) (RTV) D1 400 1.00 1.35 ± 0.241.35 ± 0.24* 0.89 ± 0.12** 1.16 ± 0.79* D2 400 1.00 0.89 ± 0.10** 0.95 ±0.15** 0.73 ± 0.19** 0.52 ± 0.23** D3 350 1.00 0.98 ± 0.12** 0.98 ±0.12** 0.65 ± 0.10** 0.34 ± 0.06** D4 300 1.00 1.12 ± 0.12 1.12 ± 0.120.79 ± 0.11* 0.55 ± 0.10* Control (NA) 1.00 1.68 ± 0.13 2.07 ± 0.16 2.76± 0.24 4.09 ± 0.53 Dosage Day 9 Day 11 Day 14 Day 16 Day 18 Groups(mg/kg) (RTV) (RTV) (RTV) (RTV) (RTV) D1 400 1.61 ± 0.98 3.18 ± 2.18* 8.88 ± 6.75 15.44 ± 11.56 20.37 ± 13.96 D2 400 0.46 ± 0.17** 0.87 ±0.35**  1.80 ± 0.69**  2.66 ± 0.88**  4.20 ± 1.02** D3 350 0.32 ± 0.05**0.37 ± 0.09**  1.00 ± 0.00**  2.17 ± 0.14**  3.16 ± 0.39** D4 300 0.55 ±0.12* 0.69 ± 0.20*  1.81 ± 0.54*  3.29 ± 0.82*  5.11 ± 1.12* Control(NA) 5.61 ± 0.83 9.22 ± 1.16 13.84 ± 2.21 17.26 ± 2.18 19.09 ± 2.45Dosage Day 21 Day 23 Day 25 Day 28 Day 30 Groups (mg/kg) (RTV) (RTV)(RTV) (RTV) (RTV) D1 400 32.34 ± 20.40 32.34 ± 20.40 33.53 ± 19.21   42± 22 51.88 ± 24.88 D2 400  8.09 ± 1.52  8.48 ± 1.37  9.62 ± 1.48 12.03 ±1.50 15.85 ± 1.92 D3 350  5.55 ± 0.66  6.21 ± 0.61  7.22 ± 0.53  9.28 ±0.73 12.93 ± 0.90 D4 300  8.73 ± 1.39  9.89 ± 1.30 11.61 ± 1.51 14.73 ±2.18 19.08 ± 2.98 Control (NA) 23.41 ± 3.10 19.77 ± 1.73 21.62 ± 1.8621.13 ± 1.62 N/A

(5) Effects of four test compounds on relative tumor proliferation rate(T/C %) of HCT-116 human colon cancer xenografts were provided. Table 9shows the effects of D1-D4 test compounds on relative tumorproliferation rate of HCT-116 human colon cancer xenografts.

TABLE 9 Effects of four cytidine derivative dimers on relative tumorproliferation rate (T/C %) of HCT-116 human colon cancer xenografts.Dosage Day 0 Day 1 Day 2 Day 4 Day 7 Groups (mg/kg) (T/C %) (T/C %) (T/C%) (T/C %) (T/C %) D1 400 0.00 80.13 65.15 32.20 28.36 D2 400 0.00 50.1540.78 25.19 16.63 D3 350 0.00 60.98 49.58 23.26 6.76 D4 300 0.00 77.0766.48 43.31 31.86 Dosage Day 9 Day 11 Day 14 Day 16 Day 18 Groups(mg/kg) (T/C %) (T/C %) (T/C %) (T/C %) (T/C %) D1 400 28.77 34.47 64.1589.47 106.69 D2 400 14.41 16.77 18.33 21.64 21.99 D3 350 4.71 3.41 5.7810.49 16.55 D4 300 30.55 25.94 32.83 37.96 26.76 Dosage Day 21 Day 23Day 25 Day 28 Day 30 Groups (mg/kg) (T/C %) (T/C %) (T/C %) (T/C %) (T/C%) D1 400 138.14 163.55 155.09 198.76 209.10 D2 400 34.57 42.87 44.5156.94 63.87 D3 350 23.70 31.41 33.40 43.93 52.09 D4 300 37.28 50.0453.68 69.72 76.91

The relative tumor proliferation rate of compound D1 group with 400mg/kg dosage reached to a minimum of 28.36% on Day 7. The relative tumorproliferation rate of compound D2 group with 400 mg/kg dosage reached toa minimum of 14.41% on Day 9. The relative tumor proliferation rate ofcompound D3 group with 350 mg/kg dosage reached to a minimum of 3.41% onDay 11. The relative tumor proliferation rate of compound D4 group with300 mg/kg dosage reached to a minimum of 25.94% on Day 11.

In the tumor inhibition experiment of the series of compounds on HCT-116human colon cancer xenografts, compounds D2, D3, D4 exhibited highertumor inhibition rates on HCT-116 human colon cancer xenografts. After aone-time intraperitoneal drug administration, there had been asignificant tumor inhibition effect without obvious influence on thebody weight of the animals, indicating a high antitumor activity of thedisclosed cytidine derivative dimers with very low toxic side effects.

As such, various embodiments provide cytidine derivative dimers(including, for example, those listed in Table 1) and/orpharmaceutically acceptable salt thereof, which can be used as effectiveingredients in therapeutic (or sometimes prophylactic) agents fortreating tumors in animals and/or humans. Accordingly, a therapeuticmethod for treating tumors using the disclosed cytidine derivativedimers and/or their salts can include: administering to a patient (e.g.,including an animal or a human) an effective amount of such therapeutic(or sometimes prophylactic) agent. For example, the therapeutic (orsometimes prophylactic) agent can be a tumor inhibiting drug. Further,methods of producing the disclosed cytidine derivative dimers and/ortheir salts are provided. Additionally, methods of producing atherapeutic or prophylactic agent including the disclosed cytidinederivative dimers and/or their salts are provided. For example, thetherapeutic or prophylactic agent including the disclosed cytidinederivative dimers and/or their salts may include the disclosedpharmaceutical composition.

Other embodiments of the disclosure will be apparent to those skilled inthe art from consideration of the specification and practice of thedisclosure disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the disclosure being indicated by the claims.

What is claimed is:
 1. A tumor inhibiting drug, comprising a cytidinederivative dimer of formula (I) or a salt form thereof,

wherein: R1 is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl, —(CH₂)_(n)-Ph, orsubstituted —(CH₂)_(n)-Ph, n being an integer from 0 to 10, and Ph beingphenyl; a carbon chain of the C₁-C₁₀ substituted alkyl is independentlysubstituted by a substituent selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup, and a combination thereof; and a carbon chain or a phenyl ring ofthe substituted —(CH₂)_(n)-Ph is independently substituted by asubstituent selected from the group consisting of halogen, cyano group,nitro group, amino group, hydroxyl group, carboxyl group, and acombination thereof; wherein: R2 is C₁-C₁₀ alkyl, C₁-C₁₀ substitutedalkyl, —(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, n being an integerfrom 0 to 10, and Ph being phenyl; a carbon chain of the C₁-C₁₀substituted alkyl is independently substituted by a substituent selectedfrom the group consisting of halogen, cyano group, nitro group, aminogroup, hydroxyl group, carboxyl group, and a combination thereof; and acarbon chain or a phenyl ring of the substituted —(CH₂)_(n)-Ph isindependently substituted by a substituent selected from the groupconsisting of halogen, cyano group, nitro group, amino group, hydroxylgroup, carboxyl group, and a combination thereof; and wherein: R3 ishydrogen, alkoxycarbonyl or substituted alkoxycarbonyl, wherein asubstituent of the substituted alkoxycarbonyl is selected from the groupconsisting of halogen, cyano group, nitro group, amino group, hydroxylgroup, carboxyl group and a combination thereof; R4 is hydrogen,alkoxycarbonyl or substituted alkoxycarbonyl, wherein a substituent ofthe substituted alkoxycarbonyl is selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup and a combination thereof; and R5 is —(CH₂)_(n)—, n being aninteger from 1 to 15; or substituted —(CH₂)_(n)— with a substituent on acarbon chain thereof, n being an integer from 1 to 15, and thesubstituent being selected from the group consisting of phenyl group,substituted phenyl group, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof; or—(CH₂)_(n)—X₁—X₂—, X₁ being O or S, X₂ being —(CH₂)_(n)-Ph-, pyrimidyl,pyranyl, imidazolyl, pyrazinyl, or pyridyl, and n being an integer from0 to
 3. 2. The tumor inhibiting drug according to claim 1, wherein R3 ishydrogen and R4 is hydrogen.
 3. The tumor inhibiting drug according toclaim 2, wherein R1 and R2 are same.
 4. The tumor inhibiting drugaccording to claim 1, wherein the tumor inhibiting drug is capable oftreating a blood cancer or a malignant solid tumor.
 5. The tumorinhibiting drug according to claim 1, wherein the tumor inhibiting drugis capable of treating a colon cancer.
 6. A method for preparing acytidine derivative dimer, comprising: preparing a compound having ageneral formula (II); and

preparing a compound having a general formula (III);

mixing the compound having the general formula (II) with sodiumcarbonate to add to a system including 1,4-dioxane and water, adding(Boc)₂O into the system for a reaction, and when the reaction ismeasured to be completed to provide a reaction product, extracting andwashing the reaction product, and then drying and concentrating to forma dried solid under a reduced pressure; adding the dried solid intochloroform, adding in pyridine and cyclic carboxylic anhydrides R₅(CO)₂Ofor an overnight reaction, concentrating to yield a viscous oil, andpurifying the viscous oil by column chromatography to obtain a compoundwith a general formula (IV):

producing the cytidine derivative dimer from the compound having thegeneral formula (IV) and the compound having the general formula (III);wherein the prepared cytidine derivative dimer has a general formula(I),

wherein: R1 is C₁-C₁₀ alkyl, C₁-C₁₀ substituted alkyl, —(CH₂)_(n)-Ph, orsubstituted —(CH₂)_(n)-Ph, n being an integer from 0 to 10, and Ph beingphenyl; a carbon chain of the C₁-C₁₀ substituted alkyl is independentlysubstituted by a substituent selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup, and a combination thereof; and a carbon chain or a phenyl ring ofthe substituted —(CH₂)_(n)-Ph is independently substituted by asubstituent selected from the group consisting of halogen, cyano group,nitro group, amino group, hydroxyl group, carboxyl group, and acombination thereof; wherein: R2 is C₁-C₁₀ alkyl, C₁-C₁₀ substitutedalkyl, —(CH₂)_(n)-Ph, or substituted —(CH₂)_(n)-Ph, n being an integerfrom 0 to 10, and Ph being phenyl; a carbon chain of the C₁-C₁₀substituted alkyl is independently substituted by a substituent selectedfrom the group consisting of halogen, cyano group, nitro group, aminogroup, hydroxyl group, carboxyl group, and a combination thereof; and acarbon chain or a phenyl ring of the substituted —(CH₂)_(n)-Ph isindependently substituted by a substituent selected from the groupconsisting of halogen, cyano group, nitro group, amino group, hydroxylgroup, carboxyl group, and a combination thereof; wherein: R3 ishydrogen, alkoxycarbonyl or substituted alkoxycarbonyl, and asubstituent of the substituted alkoxycarbonyl is selected from the groupconsisting of halogen, cyano group, nitro group, amino group, hydroxylgroup, carboxyl group and a combination thereof; R4 is hydrogen,alkoxycarbonyl or substituted alkoxycarbonyl, and a substituent of thesubstituted alkoxycarbonyl is selected from the group consisting ofhalogen, cyano group, nitro group, amino group, hydroxyl group, carboxylgroup and a combination thereof; and wherein: R5 is —(CH₂)_(n)—, n beingan integer from 1 to 15; or substituted —(CH₂)_(n)— with a substituenton a carbon chain thereof, n being an integer from 1 to 15, and thesubstituent being selected from the group consisting of phenyl group,substituted phenyl group, cyano group, nitro group, amino group,hydroxyl group, carboxyl group, and a combination thereof; or—(CH₂)_(n)—X₁—X₂—, X₁ being O or S, X₂ being —(CH₂)_(n)-Ph-, pyrimidyl,pyranyl, imidazolyl, pyrazinyl, or pyridyl, and n being an integer from0 to
 3. 7. The method according to claim 6, further comprising: mixingand adding the compound having the general formula (IV), the compoundhaving the general formula (III), and N,N′-dicyclohexylcarbodiimide(DCC) to methylene chloride, adding in 4-dimethylaminopyridine (DMAP)for a reaction, and when the reaction is measured to be completed,washing, drying, and concentrating the reaction mixture to form a driedsolid under a reduced pressure, wherein trifluoroacetic acid (TFA) anddichloromethane (DCM) are added to the dried solid, stirred at roomtemperature, cooled in an ice bath, and filtered to provide a whitesolid, and wherein the white solid is concentrated into a viscous oil,purified by column chromatography to produce the cytidine derivativedimer.